Activation of nuclear factor kappaB in radioresistance of TP53-inactive human keratinocytes.

Many tumors show a mutant or inactive tumor suppressor p53 (TP53) status, and TP53 in the tumor-carrying human papillomavirus (HPV) may be dysfunctional because of inhibition by the viral protein HPV E6. Molecular mechanisms underlying radiation responses and the radiation-induced resistant phenotype in the TP53-inactive tumor have not been well investigated. In the present study, using a human keratinocyte line (HK18) with TP53 inhibited by HPV18 infection, we demonstrated that nuclear factor (NF)-kappaB is responsible for a major portion of the radioresistance observed in a cell population (HK18-IR) derived from HK18 cells by fractionated ionizing radiation (FIR; 2 Gy/fraction; total dose, 60 Gy). HK18-IR cells showed increased clonogenic radioresistance [dose-modifying factor (DMF), 1.47], reduced apoptotic response, and a shortened radiation-induced growth delay. Both DNA-binding and reporter transcriptional activity of NF-kappaB, but not of TP53, were activated in HK18-IR cells compared with the parental HK18 cells; this activation was observed both before and after a single dose of 5 Gy. To determine target genes responsive to NF-kappaB activation, DNA microarray profiles for 588 genes were matched in HK18-IR cells compared with those in HK18 cells; the paired comparisons were made for basal levels before irradiation or for levels 24 h after 5 Gy. For 25 genes, a 2- to 5-fold up-regulation in HK18-IR cells relative to HK18 cells was similar when comparisons were made for basal levels or for levels after irradiation. Included in the approximately 4% of genes activated in HK18-IR cells, were six genes (Cyclin B1, Cyclin D1, HIAP, BAG-1, TTF, and fibronectin) putatively linked to NF-kappaB regulation. We then measured the expression of this group of FIR-regulated genes in HK18-IR cells expressing a dominant-negative mutant IkappaB (mIkappaB) that inhibited NF-kappaB activation. Clonogenic radioresistance was reduced greatly in the mIkappaB transfectants (DMF, 1.18 and 1.10, respectively, at 10% and 1% of isosurvival for mIkappaB transfectants compared with 1.47 and 1.45, respectively, for vector control transfectants). Expressions of Cyclin B1, Cyclin D1, and HIAP were down-regulated by the inhibition of NF-kappaB. These results suggest that transcription of NF-kappaB and a group of NF-kappaB target genes are involved in radioresistance in FIR-treated tumor cells with inactive TP53.